Johns Hopkins University
Animal Care and Use Committee
Euthanasia of Rats and Mice Using Carbon Dioxide1
PURPOSE: This document provides guidance on the correct procedures to follow when
euthanizing rodents using carbon dioxide.
BACKGROUND: Euthanasia of animals at Johns Hopkins University must be carried out according
to the most recent guidance of the American Veterinary Medical Association (AVMA).2 Carbon
dioxide (CO2) inhalation is a common method of euthanasia used for rats and mice. It is the method
that will be used by central facilities staff with rats and mice identified for euthanasia by
researchers. Use of CO2 euthanasia by researchers must be included in an ACUC-approved protocol.
Appropriate technique, equipment, and source of CO2 must be used. Compressed CO2 gas in
cylinders is the only approved source because the inflow of gas to the euthanasia chamber can be
regulated precisely. To insure the CO2 flow rate displaces 10% - 30% of the chamber volume per
minute a flow meter must be attached to the regulator which is set at 50psi. CO2 generated by
other means such as dry ice, fire extinguishers, or chemical means (e.g., antacids) is
unacceptable.
Upon completion of the procedure, death must be confirmed for each animal by one or more of the
methods listed below. CO2 is an anesthetic and failure of the animal to move or show a reflex
response is not sufficient to confirm death. Disposal of an anesthetized, rather than a
euthanized, animal is a serious animal welfare concern and a significant breach of the
conditions of JHU’s approved Public Health Service Animal Welfare Assurance.
Understanding how to avoid this is incumbent upon anyone carrying out euthanasia with CO2.
PROCEDURE: Animals in the euthanasia chamber should be readily visible. Do not overcrowd the
chamber by combining multiple cages. All animals in the chamber must be able to make normal
postural adjustments.
Sudden exposure of conscious animals to CO2 concentrations of 70% or greater has been shown to
be distressful3. Yet death should be induced as quickly as possible. Without pre-charging the
chamber, place the animals in the chamber and introduce 100% CO2 at 10% - 30% of the chamber
volume per minute so as to optimize reduction in distress. Since this is critical to the humane
application of CO2, it is necessary to have a pressure-reducing regulator and flow meter that will
Approved by the JHU Animal Care and Use Committee on: April 17, 2003---revised 9/28/2006, 7/17/08,
1/15/15
2
AVMA Guidelines on Euthanasia of Animals: 2013 ed. Available at www.jhu.edu/animalcare.
3 Dannenman, P. J., Stein, S., Walshaw, S. O. Humane and practical implications of using carbon dioxide mixed
with oxygen for anesthesia or euthanasia of rats. Laboratory Animal Science, 1997, 47: 376-385.
1
generate the recommended displacement rates. Animals should be exposed to the CO2 for the
duration listed in the table below. Because the acceptable flow rate is a range and size of the
chamber will affect the exposure time, the table lists a range of times.
Age of Animal
Time gas flow
Time continued exposure
14 day to adult mice
14 day to adult rats
Newborn to 14 day mouse
or rat pups
2 - 4 minutes
2 - 4 minutes
2 - 4 minutes
3 - 6 minutes
5 - 10 minutes
5 - 10 minutes
Total time
exposure
5 - 10 minutes
7 - 14 minutes
7 - 14 minutes
Neonatal animals (up to 14 days of age) are resistant to the effects of CO2 and therefore it is
recommended that a secondary physical method follow the CO2 exposure. This may include
decapitation using a sharp blade or cervical dislocation.
Death must be ensured for all animals prior to disposal. One of the methods below must be used
unless harvest of vital organs will occur right away. All the methods listed below are approved by
the Animal Care and Use Committee, and may be used even if they were not included in your
approved protocol.
1. Verification that the heart has stopped beating and the animal is not breathing; paleness of
mucosal tissue. (This typically is not very practical if many animals are being sacrificed at
one time since it involves handling each one.)
2. Observation that the animal fails to recover within 10 minutes after CO2 exposure ends.
3. Cervical dislocation.
4. Thoracotomy.
5. Decapitation.
.